Differential Assessment of Factor Xa Activity and Global Blood Coagulability Utilizing Novel Dielectric Coagulometry.

An easy-to-use assessment for activated factor X (FXa) is lacking despite its pivotal role in the coagulation. Dielectric blood coagulometry (DBCM) was recently invented as a novel assessment tool for determining the whole blood coagulability by measuring the temporal change in the permittivity of blood. We previously reported that it could evaluate the global blood coagulability. This study aimed to apply the DBCM for assessing FXa activity and its inhibition by anticoagulants. We performed the DBCM analysis along with measurement of the FXa activity by a fluorometric assay in samples from healthy subjects, and identified a new index named maximum acceleration time (MAT) that had a correlation to the FXa activity. Next the DBCM analysis was performed using blood samples mixed with anticoagulants (unfractionated heparin, dalteparin, and edoxaban). Blood samples with three anticoagulants had different profiles of the temporal change in the permittivity, reflecting their different selectivity for FXa. We compared the MAT with the anti-FXa activity assay, and found that the prolongation of MAT was similarly correlated with the anti-FXa activity regardless of the type of anticoagulants. In conclusion, the DBCM has the possibility for evaluating the innate FXa activity and effect of anticoagulants focusing on their FXa inhibition.

New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx.

OBJECTIVE: It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators' ability to assess the role of the glycocalyx in vascular health. APPROACH AND RESULTS: We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface's functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm(2) on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography-mass spectrometry analysis. CONCLUSIONS: Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Qualitative and quantitative pharmacophore-similarity assessment of anthranilamide-based factor Xa inhibitors: applications on similar molecules with identical biological endpoints.

It is a conventional practice to exclude molecules with identical biological endpoints to avoid bias in the resulting hypothesis model. Despite the diverse chemical functionalities, the receptor interactions of such molecules are often unexplored. The present study motivates the selection of these molecules diversified by single atom or functional group compared to internal molecules as external set and helps in the understanding of corresponding effects toward receptor interactions and biological endpoints. Applied on anthranilamide-series of factor Xa analogs, the inhibitory activities were correlated (r(2) = 0.99) and validated (q(2) = 0.68) with distance-based pharmacophore descriptors using support vector machine. The selected external set molecules exhibited better prediction accuracy by securing activities less than one residual threshold. The effect on inhibitory activity was assessed by the examination of pharmacophore-similarity and its interactions with key residues of Human factor Xa enzyme using molecular docking approach. Furthermore, qualitative pharmacophore models were developed on the subset of molecular dataset divided as most actives, moderately actives and least actives, to recognize crucial activity governing pharmacophore features. The outcome of this study will bring new insights about the requirements of pharmacophore features and prioritizes its selection in the design and optimization of potent Xa inhibitors.

At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.

Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease bioactivity fingerprints in biological studies on venoms.

Anti-coagulation assessment with prothrombin time and anti-Xa assays in real-world patients on treatment with rivaroxaban.

Monitoring of anti-coagulation with the direct factor Xa inhibitor rivaroxaban is considered unnecessary in a routine clinical setting. However, assessment of its anti-coagulant effect may be desirable in certain clinical situations. We assessed prothrombin time (PT) reagents and commercially available anti-Xa assays (Biophen) calibrated for rivaroxaban and heparin in comparison to liquid chromatography-mass spectrometry (LC-MS/MS) measurements of rivaroxaban concentration in samples from patients on treatment with rivaroxaban for stroke prevention in atrial fibrillation. Citrate plasma samples were obtained from 30 randomly selected patients on uninterrupted treatment with rivaroxaban for a minimum of 1 month. The anti-Xa assays, direct Xa inhibitor (DiXa-I®), and Heparin LRT® were conducted for both wide and low calibrations for rivaroxaban. Measurements were compared to LC-MS/MS using correlation, linear regression, intra-class correlation, and Bland-Altman analysis. In 30 patients (9 female) of median age 71.5 years and BMI 26.5 kg/m(2), rivaroxaban concentrations between 2.4 and 625 ng/ml (median 82 ng/ml) were measured by LC-MS/MS. PT reagents were poorly correlated with rivaroxaban concentrations (r (2) = 0.52 and 0.09). Anti-Xa assays DiXa-I (r (2) = 0.95) and Heparin LRT (r (2) = 0.97) were correlated with rivaroxaban in all concentrations, but especially in low concentrations with low calibrations (r (2) = 0.97 and 0.98, respectively). The highest agreement occurred between Heparin LRT and low rivaroxaban concentrations with a mean difference of -5.3 ng/ml (limits of agreement, 12.9 to 2.4 ng/ml). Anti-Xa assays can indirectly determine the concentration of rivaroxaban for a wide range of concentrations in real-world patients. An interpretation of anti-Xa and PT measurements in treatment with rivaroxaban requires knowledge of the local reagents.

Assessment of anti-factor Xa activity of heparin in binary parenteral nutrition admixtures for premature neonates.

An in vitro study was carried out to determine the anti-Xa activity of heparin in binary parenteral nutrition (BPN) admixtures for premature neonates in our neonatal intensive care unit (NICU) after a 24-hour infusion, as well as to assess drug interaction with a 50% glucose solution. Two types of bags were prepared: (1) BPN admixtures (composition defined in the NICU) including sodium heparin at 77 UI/mL and (2) bags containing only G50% with sodium heparin at 193 UI/mL. The anti-Xa activity of heparin was measured in bags at T0, after the 24-hour infusion and in eluates at the outlet of the infusion line after 24hours, using a validated chromogenic anti-Xa method. Comparisons of the mean concentration observed with the theoretical value for anti-Xa activity were performed with the Student t-test. Mean values of anti-Xa activity do not differ significantly from the values expected for all conditions. We found a slight variation in anti-Xa activity when infused over 24hours for both types of bags, with and without in-line filtration, showing that heparin remains stable during this infusion period in both BPN admixtures and G50%.

Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B.

In this study, biochemical and pharmacological characterization of Brevithrombolase, a fibrinolytic serine protease purified from Brevibacillus brevis strain FF02B has been reported. An assessment of its thrombolytic potency has also been made. The molecular mass of this monomeric protease was determined as 55 kDa, and 56043 Da, respectively, by SDS-PAGE and MALDI-TOF-MS. In the analytical studies, the N-terminal sequence of Brevithrombolase was found to be blocked; however, the peptide mass fingerprinting and amino acid composition analyses demonstrated the similarity of Brevithrombolase with endopeptidases in possessing serine in their catalytic triad. This finding was confirmed by the observation that the serine protease inhibitors decrease the catalytic (fibrinolytic) activity of Brevithrombolase. The secondary structure of Brevithrombolase was composed of 30.6% alpha helix and 69.4% random coil. Brevithrombolase showed the Km and Vmax values towards the chromogenic substrate for plasmin at 0.39 mM and 14.3 µmol/min, respectively. Brevithrombolase demonstrated optimum fibrinolytic activity at pH 7.4 and 37 °C, and showed marginal hydrolytic activity towards globulin, casein and fibrinogen. The anticoagulant potency of Brevithrombolase was comparable to the low molecular mass heparin/antithrombin-III and warfarin. Among the three enzymes-Brevithrombolase, plasmin and streptokinase-the fibrinolytic activity and in vitro thrombolytic potency of Brevithrombolase was found to be superior. The RP-HPLC and SDS-PAGE analyses suggested a similar pattern of fibrin degradation by Brevithrombolase and plasmin, indicating that former enzyme is a plasmin-like fibrinolytic serine protease. Brevithrombolase did not show in vitro cytotoxicity on HT29 and HeLa cells or hemolytic activity. At a dose of 10 mg/kg, Brevithrombolase did not exhibit lethality or toxicity on Wistar strain albino rats. Brevithrombolase did not inhibit factor Xa, and its mechanism of anticoagulant action was associated with the enzymatic cleavage of thrombin. The combined properties of Brevithrombolase indicate its therapeutic potential in peptide-based cardiovascular drug development.

A randomised assessment of the pharmacokinetic, pharmacodynamic and safety interaction between apixaban and enoxaparin in healthy subjects.

Following major orthopaedic surgery, guidelines usually recommend continued thromboprophylaxis after hospitalisation. The availability of an effective oral anticoagulant with an acceptable safety profile that does not require routine clinical monitoring may lead clinicians to switch patients from subcutaneous to an oral therapy either during hospitalisation or at discharge. The purpose of this study was to assess the effect of enoxaparin on the pharmacokinetics, pharmacodynamics and safety of apixaban, an oral, direct inhibitor of coagulation factor Xa. In this four-period, crossover study, 20 healthy subjects were randomised to receive single doses of apixaban 5 mg orally; enoxaparin 40 mg subcutaneously; apixaban 5 mg and enoxaparin 40 mg concomitantly; and apixaban 5 mg followed 6 hours (h) after by enoxaparin 40 mg. Pharmacokinetics of apixaban were not affected by enoxaparin. Average peak pharmacodynamic effect, measured by anti-Xa activity, was 1.36 U/ml after administration of apixaban and was 0.42 U/ml after enoxaparin. Following co-administration of apixaban and enoxaparin, peak anti-Xa activity was 42% higher than for apixaban alone. Following administration of enoxaparin 6 h after apixaban, peak anti-Xa activity was 15% higher than for apixaban alone. In conclusion, enoxaparin had no effect on the pharmacokinetics of apixaban. The increase in anti-Xa activity after co-administration was modest and appeared to be additive. Peak anti-Xa activity increases are mitigated by separating administration of subcutaneous anticoagulation and apixaban when switching between therapies; the potential for pharmacodynamic interaction may be further mitigated by transitioning at the next scheduled dose (12 h).

The effects of urinary trypsin inhibitor on liver function and inflammatory factors in patients undergoing hepatectomy: a prospective, randomized, controlled clinical study.

BACKGROUND: The inhibition of inflammation exerts benefits following massive hepatectomy in animals but not in the clinic. The aim of this study was to investigate the effectiveness and mechanism of ulinastatin on liver function and outcomes following hepatectomy. METHODS: One hundred seventy-six patients undergoing hepatectomy were randomized into the treatment group (n = 86) and the control group (n = 90), receiving ulinastatin 150,000 U twice daily for 3 days and saline vehicle, respectively. Liver function, coagulation, thrombokinase, lymphocyte subsets CD4 and CD8, C-reactive protein, inducible nitric oxide synthase, and cytokines were measured. Clinical outcomes were also evaluated. RESULTS: Serum alanine transaminase, aspartate transferase, inducible nitric oxide synthase, and tumor necrosis factor-α levels were significantly lower after ulinastatin treatment, and the response of bilirubin was delayed. The benefits of ulinastatin were shown mainly in major hepatectomy earlier after surgery. The treatment significantly reduced hospital length of stay and recovery-related cost. CONCLUSIONS: Ulinastatin protects liver function and improves clinical outcomes, possibly via the inhibition of inflammation and oxidation at an earlier stage following major hepatectomy.

Safety assessment and pharmacodynamics of a novel ultra low molecular weight heparin (RO-14) in healthy volunteers--a first-time-in-human single ascending dose study.

INTRODUCTION: RO-14 is a novel ultra low molecular heparin. The purpose of this study was to evaluate the safety and pharmacodynamic profile of RO-14 in healthy males. MATERIALS AND METHODS: We conducted a two-stage, single-center, open-label, randomized study. Two cohorts of 6 volunteers were randomly assigned to 12 single, ascending subcutaneous doses (1750-19950IU of anti-FXa activity) in an alternating crossover fashion. Safety was assessed by spontaneous/elicited adverse events, medical examination and laboratory tests. Anti-FXa activity and anti-FIIa activity were assessed throughout the 24hours after dosing. Dose proportionality and linearity of the anti-FXa activity were evaluated. RESULTS: All doses were well tolerated and there were no bleeding events. At the lowest dose, anti-FXa activity A(max) was 0.16 (±0.02) IU/mL and AUC(0-24) was 1.11 (±0.24) IU*h/mL, At the highest dose anti-FXa activity A(max) was 1.67 (±0.15) IU/mL; AUC(0-24) was 21.48 (±4.46) IU*h/mL and t½ was 8.05h. Mean T(max) (all doses) was 2.86 (±0.39) h. RO-14 showed proportional and linear pharmacodynamics [normalized A(max) among doses (p=0.594) and normalized AUC(0-24) (p=0.092), correlations between A(max-)dose (R(2)=0.89, p<0.001) and AUC(0-24)-dose (R(2)=0.86, p<0.001)]. Anti-FIIa activity was below the detection limit (0.1IU/ml) at all dose levels. No clinically significant changes were observed in the platelet count, APTT, PT, TT, fibrinogen and antithrombin. CONCLUSIONS: In this phase I study, RO-14 exhibited a good safety profile, anti-FXa activity for either prophylaxis or treatment of venous thromboembolism, linear pharmacodynamics, a longer elimination half-life than currently marketed low molecular weight heparin and no anti-FIIa activity.

Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins.

There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay option - indirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD ≤ 0.5%, LOQ ≤ 0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.

Assessment of the heparin-binding AN69 ST hemodialysis membrane: II. Clinical studies without heparin administration.

Binding polyanionic unfractionated heparin over the modified AN69 polyacrylonitrile membrane, the surface electronegativity of which has been neutralized by polyethyleneimine (AN69-ST), renders the membrane more hemocompatible. This property was tested in two groups of long-term hemodialysis patients. Results were rated as massive or partial clotting of a dialyzer at the end of the session. Group I patients were included in a prospective, cross-over study comparing standard dialysis with hemodialysis without systemic administration of unfractionated heparin (n = 12, 123 sessions). In all instances, priming was made with 2 I saline containing 5,000 IU/l heparin. Only patchy or partial clotting was observed in 11% and 39% of the sessions with standard and heparin-free administration, respectively. Group II patients were included in an open, observational pilot study testing the effects of the heparin-coated membrane, without systemic administration of heparin, in patients at high risk of bleeding (n = 68, 331 sessions). Massive clotting was observed in six sessions only (less than 2%) and normal or slightly patchy dialyzers were found in 88% of the sessions. It is concluded that the dialysis AN69 ST membrane, after adequate priming at bedside, can be used without systemic administration of heparin for hemodialysis in patients at high risk of bleeding.

Assessment of heparin binding to the AN69 ST hemodialysis membrane: I. Preclinical studies.

The AN69 ST membrane was designed to render the surface of the native polyacrylonitrile polymer less cationic. This was achieved by layering the membrane with the polycationic biopolymer polyethyleneimine. This new membrane is able to bind heparin to its surface, through electrical interactions, without altering the reactivity of the sulfonate groups of the membrane, regularly distributed in the membrane bulk. The kinetics of unfractionated or low-molecular-weight heparins were studied in vitro and in vivo in sheep. Encouraging results were obtained indicating that heparin-coated hemodialyzers are potent anticoagulants. Priming the AN69 ST membrane-equipped hemodialyzer with heparin, as in regular hemodialysis, could allow drastic reduction of heparin consumption in hemodialysis.

Pharmacodynamic markers in the early clinical assessment of otamixaban, a direct factor Xa inhibitor.

This manuscript reports the assessment of pharmacodynamic (PD) markers of anti-coagulation in the first-in-man study with the novel direct Factor Xa (FXa) inhibitor, otamixaban, with a brief description of safety and pharmacokinetic (PK) findings. The study comprised ten consecutive parallel groups of healthy male subjects (6 active, 2 placebo per group). Eight groups received escalating intravenous doses of otamixaban as 6-hour infusions (1.7 to 183 microg/kg/h) and two groups received a bolus dose (30 or 120 microg/kg) with a 6-hour infusion (60 or 140 microg/ kg/h, respectively). PD markers included anti-FXa activity and clotting time measurements, i.e. activated Thromboplastin Time (aPTT), Prothrombin Time (PT), Heptest Clotting Time (HCT), and Russell's Viper Venom-induced clotting Time (RVVT). In addition, Endogenous Thrombin Potential (ETP) was assessed in the bolus-plus-infusion dose groups. Otamixaban was well tolerated. Otamixaban plasma concentrations increased with escalating dose, were maximal at the end-of-infusion (C(eoi)), and decreased rapidly as the infusion was stopped. Anti-FXa activity coincided with otamixaban plasma concentrations and clotting time measurements followed the same pattern. Maximal changes from baseline at C(eoi) were 1.9 +/- 0.2 for aPTT, 2.0 +/- 0.2 for PT, 5.1 +/- 0.6 for HCT, and 4.5 +/- 1.2 for RVVT. Otamixaban inhibited thrombin generation (24% decrease in ETP) and a delay in thrombin generation was noticed in vitro at high concentrations.

Understanding protein-ligand interactions: the price of protein flexibility.

In order to design selective, high-affinity ligands to a target protein, it is advantageous to understand the structural determinants for protein-ligand complex formation at the atomic level. In a model system, we have successively mapped the factor Xa binding site onto trypsin, showing that certain mutations influence both protein structure and inhibitor specificity. Our previous studies have shown that introduction of the 172SSFI175 sequence of factor Xa into rat or bovine trypsin results in the destabilisation of the intermediate helix with burial of Phe174 (the down conformation). Surface exposure of the latter residue (the up conformation) is critical for the correct formation of the aromatic box found in factor Xa-ligand complexes. In the present study, we investigate the influence of aromatic residues in position 174. Replacement with the bulky tryptophan (SSWI) shows reduced affinity for benzamidine-based inhibitors (1) and (4), whereas removal of the side-chain (alanine, SSAI) or exchange with a hydrophilic residue (arginine, SSRI) leads to a significant loss in affinity for all inhibitors studied. The variants could be crystallised in the presence of different inhibitors in multiple crystal forms. Structural characterisation of the variants revealed three different conformations of the intermediate helix and 175 loop in SSAI (down, up and super-up), as well as a complete disorder of this region in one crystal form of SSRI, suggesting that the compromised affinity of these variants is related to conformational flexibility. The influence of Glu217, peripheral to the ligand-binding site in factor Xa, was investigated. Introduction of Glu217 into trypsin variants containing the SSFI sequence exhibited enhanced affinity for the factor Xa ligands (2) and (3). The crystal structures of these variants also exhibited the down and super-up conformations, the latter of which could be converted to up upon soaking and binding of inhibitor (2). The improved affinity of the Glu217-containing variants appears to be due to a shift towards the up conformation. Thus, the reduction in affinity caused by conformational variability of the protein target can be partially or wholly offset by compensatory binding to the up conformation. The insights provided by these studies will be helpful in improving our understanding of ligand binding for the drug design process.

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.